Hematopoiesis is a complex program of cellular differentiation. At the center of this system is a population of hematopoietic stem cells (HSCs) endowed with the ability to self-renew as well as to differentiate into mature blood and immune cell types. Recent evidence also suggests that akin to the normal hematopoietic system, blood cancers (leukemias) are organized in a similar hierarchical fashion with long-lived leukemic stem cells (LSC) at their apex. Despite advances in the purification and functional characterization of normal and malignant stem cells, our understanding of how individual HSC/LSCs behave in vivo, is still very poor. Insight into the number and the contribution of HSCs to the formation of blood lineages would have great significance for the design of therapeutic interventions such as gene therapy and transplantation. Although some knowledge of this process has been gained through classic retroviral marking studies, all of those studies involved ex-vivo manipulation of HSCs and their transplantation into lethally-irradiated recipients, clearly non-physiological conditions. In this exploratory proposal, we seek to develop a novel strategy which will allow us to uniquely tag and follow the progeny of HSCs and LSCs in situ. Utilizing a combination of the tetracycline-inducible and the sleeping beauty (SB) transposase systems, we will create a murine model in which HSCs can be marked while still in the bone marrow and therefore follow their behavior under completely normal physiological conditions. It is proposed, firstly, to characterize transposon marking at the level of the HSC. We will determine how many HSCs can be uniquely marked and whether each individual HSC has a unique transposition tag. Secondly, once the marking parameters are defined, we will study the dynamic behavior of hematopoietic stem cell clones in situ. After induction of transposition we will determine how many HSC clones contribute to multi-lineage hematopoiesis in steady state hematopoiesis, and what the mechanisms of this contribution are. In the third part, we will study the fluctuation of clonal contribution in leukemia model. This study will allow us for the first time to define the behavior of HSCs in steady state hematopoiesis. If successful, this novel system could be used to study stem cell dynamics in potentially every tissue and type of tumor. PROJECT NARRATIVE: This proposal will allow us to understand how blood-forming stem cells function in an organism. Therefore our results will allow us to understand how stem cell function is affected in clinical conditions such as bone marrow failure syndromes and cancers of the blood.